Abstract

G-protein-coupled receptor activation is generally analyzed under equilibrium conditions. However, real-life receptor functions are often dependent on very short, transient stimuli that may not allow the achievement of a steady state. This is particularly true for synaptic receptors such as the α2A-adrenergic receptor (α2A-AR). Therefore, we developed a fluorescence resonance energy transfer-based technology to study nonequilibrium α2A-AR function in living cells. To examine the effects of increasing concentrations of the endogenous agonist norepinephrine on the speed and extent of α2A-AR activation with very high temporal resolution, we took advantage of a fluorophore-containing α2A-AR sensor. The results indicated that the efficacy of norepinephrine in eliciting receptor activation increased in a time-dependent way, reaching the maximum with a half-life of ∼60 ms. The EC50 values under nonequilibrium conditions start at ∼26 μM (at 40 ms) and show a 10-fold decrease until the steady state is achieved. To analyze the ability of norepinephrine to trigger a downstream intracellular response after α2A-AR stimulation, we monitored the kinetics and amplitude of Gi activation in real time by using a fluorophore-containing Gi sensor. The results show that both the efficacy and the potency of norepinephrine in inducing Gi activation achieve a steady state more slowly, compared with receptor activation, and that the initial EC50 value of ∼100 nM decreases in an exponential way, reaching the minimal value of ∼10 nM at equilibrium. Therefore, both the efficacy and the potency of norepinephrine increase ∼10-fold over a few seconds of agonist stimulation, which illustrates that receptor and G-protein signaling and signal amplification are highly time-dependent phenomena.

Footnotes

This work was supported by the Deutsche Forschungsgemeinschaft [Grant SFB487] and the European Research Council [Advanced Grant “Topas”].